Glycosphingolipid (GSL)-enriched rafts and caveolae are membrane microdomains that putatively function as lateral organizing zones for signaling proteins involved in oncogenesis and as targeted sites by bacteria, their toxins, and envelope viruses. The processes by which GSL-enriched domains are formed and maintained are not well understood and may involve proteins that bind and transfer GSLs between cell membranes. Our long-range goal is to understand how the structural features of GSLs govern their lateral mixing behavior with other membrane lipids and to identify/characterize the physiochemical features within the GSL-enriched membrane environment that modulate interactions with lipid transfer proteins (GLTPs) that selectively transfer GSLs between membranes. The central hypothesis is that the physical environment produced by lipid compositional changes and the resulting changes in lateral organizational state of GSLs regulate GLTP translocation on and off the membrane, thereby controlling GLTP activity. The hypothesis will be tested by pursuing 4 specific aims: 1) Define the mechanism of GLTP action and determine how GSL-lipid packing interactions with lamellar surfaces regulate GTLP activity;2) Map the membrane interaction region of human GLTP by site-directed mutagenesis;3) Identify the structural features of GSLs that modulate their mixing interactions with phospholipids and sterols, and define the physical nature of the lamellar environment that is produced by GSL-lipid interactions within and across the bilayer;4) Ascertain the mechanism of action used by fungal and plant GLTP orthologs (HET-C2 &ACD11) and determine the functional relationship of human GLTP1 to 2 new human homologs (GLTP2 &GLTP3). 5) Investigate potential GLTP cell biological functions involving regulation of GSLs expression, stabilization of raft microdomains and signaling pathways linked to apoptosis. The objectives will be achieved by synthetically manipulating GSL structure, and by using complementary monolayer and bilayer model membrane systems to study GSL-lipid and membrane-protein interactions, in concert with functional and point mutational analyses of GLTPs. The proposed work is innovative because it capitalizes on the first-ever, structural insights into human GLTP. The novel 2-layer, all alpha helical topology of GLTP1 is unique compared to other known lipid binding transfer proteins, suggesting that the GLTP folding motif defines a novel protein family. It is our expectation that the proposed studies of GSLs, human GLTPs and related orthologs will provide unparalleled insights into the workings of this emerging new protein superfamily. This new knowledge is expected to be significant by providing a foundation for using GLTP in new and innovative ways, such as introducing specific GSL antigens into cancer cells to help achieve targeted destruction of diseased cells via immunotherapeutic and site-directed toxicological means.